Chlamydia is one of the most important infectious agents from a public health perspective. In 2011 more than 1.4 million cases of chlamydial infections were reported to the CDC making it the most commonly reported infectious disease in the U.S. Chlamydia causes an intracellular infection that is unusual among pathogenic bacteria because it involves conversion between two specialized forms of the bacterium. The reticulate body (RB) is an intracellular form that replicates by binary fission, while the elementary body (EB) is the infectious form that can transmit the infection to a new cell. A successful infectious cycle involves both RB replication and RB-to-EB conversion within an intracellular compartment called the chlamydial inclusion, but it is not known how these processes are regulated. We have developed an innovative approach to obtain detailed three dimensional views of a Chlamydia-infected cell. We first perform electron microscopy imaging on serial sections through the cell, then use computational methods to reconstruct these scans into a 3D image and finally trace the outline of each of the bacteria. With this approach we can visualize the entire chlamydial inclusion and the numbers and locations of all the RBs and EBs. In Aim 1 of this application, we propose to obtain and compare 3D reconstructions of Chlamydia-infected cells over the course of the 48-hour developmental cycle. In Aim 2, we will analyze this data with mathematical and modeling approaches to determine if the proportion of RBs that are replicating or converting changes with time. We will also examine if RB replication and RB-to-EB conversion correlate with external factors such as the size of the inclusion or the surface area of the inclusion membrane. This novel approach will provide an unprecedented level of quantitative and spatial detail about a Chlamydia-infected cell. This information will help us to understand fundamental properties about the intracellular infection, including how this important pathogen replicates and produces infectious progeny inside a human cell.